The objective of this proposal is to study the regulation of genomic imprinting in mouse using the maternally expressed H19 gene as the experimental system. Genomic imprinting is the differential expression of alleles dependent upon the parental origin. It occurs in mammals and, as a consequence, both a maternal and a paternal copy of certain genes are required for normal development. In addition, imprinting is implicated in several human diseases, and the understanding of imprinting may facilitate the correction of these human diseases. Although several genes have been shown to be imprinted in both mice and humans, the mechanism by which parental identity is assigned is unclear. However, a strong candidate for the differential marking of alleles is methylation of cytosine residues in CpG dinucleotides. For H19, a paternal specific methylated sequence has been identified which is not required for transcription of H19. The specific aim of this proposal is to determine the role of the paternally methylated H19 sequence by removing the sequence from the endogenous locus using homologous recombination and then studying the outcome in mice by analyzing H19 expression from the disrnpted allele in a parental-specific manner.